The phenotype of single normal cells is in the human body is defined by the set of genes that are expressed at a certain time point. These gene expression patterns define the combination and individual levels of proteins that provide structure and function to all living cells and at a level of higher complexity all organs. Differentiation processes that are essential for all organs to function appropriately are also regulated by shifts in the level and combination of individual genes. Differential gene expression patterns determine the status of a cell for example as a stem cell that give rise to progenies that can undergo differentiation to fully differentiated cells, with substantially changed gene expression patterns. These finally conclude their lifespan by again changing their gene expression pattern and thereby adopting properties of aging and finally dying cells. Although the molecular control of these processes are far from being solved several lines of evidence suggest that substantial parts of this control is mediated by distinct methylation of certain GpC-islands within the human genome.
Conceptually the addition of methyl-(C(H)3)-groups to the certain nucleotides within the cell's genome results in a condensed and tightened structure of the methylated stretch of the genome and consequently inhibition of its transcription. Thus, heavily methylated parts of the genome are usually not transcribed into mRNA (silenced). This mechanism is an important part of gene regulation and imprinting and one of the central biological features that determine the critical steps in a living cell (Collas et al., 2007). The detail impact of methylation in differentiation processes has only very superficially been investigated so far (Kiefer, 2007). However, more detailed insight has been gathered during the transformation processes that are involved in the transformation of a normal cell into a full blown neoplastic cancer cell (Esteller, 2007; Feinberg, 2004). Particularly for genes with important tumor suppressive activities methylation of the promoter regions have been observed that resulted in reduced expression of the respective genes in the emerging cancer cells. Hence methylation is one of the central molecular steps in the inactivation of tumor suppressor gene besides deletion and/or mutation and has been clearly been proven for many tumor suppressive genes as for example the cyclin dependent kinase inhibitor p16INK4a, the DNA mismatch repair genes MSH2, MHL1, and others (Esteller, 2007; Feinberg, 2004).
Besides its impact on the chromatin structure, more refined methylation of distinct nucleotides may also affect the binding properties of certain transcription factors (Klose and Bird, 2006). It thus may influence the affinity of positive or negative acting transcription factors and thus modifies the action and activity of the transcription factors which again results in either more active or reduced expression of the respective genes.
Human papillomaviruses are important epitheliotropic viruses that cause epithelial proliferations of the skin or mucosal surfaces. They attracted particular scientific interest when it became clear, that certain distinct types (high risk papillomaviruses, HR-HPVs) are associated with neoplastic lesions particularly of mucosal surfaces of the genital tract and most importantly of the uterine cervix (zur Hausen, 2002). It is generally accepted today that persistent infections of HR-HPV types (particularly HPV 16 and 18) are an essential prerequisite for the development of cervical cancer. Certain viral genes referred to as E6 and E7 were shown to be continuously expressed in cervical and other HPV-associated cancer cells. They further were shown to induce neoplastic transformation in in vitro cultured primary human epithelial cells and importantly, their expression is essentially required to induce and maintain the neoplastic growth features of cervical carcinoma cells or their high grade precursors.
Up to now, about 13 high risk HPV types have been characterized (Munoz et al., 2003). These viruses are commonly found also in women who have (not yet) developed any clinically detectable lesions. The peak incidence of the virus infections is found in young women at the ages of 15 to 30 years of life, whereas it declines in women of older age (Schiffman et al., 2007). This clearly points to the sexually transmitted characteristics of HR-HPV-infections that are also found in young men at comparable frequencies. The major difference between both genders however is that in males HR-HPV infections usually induce subclinical infections that are rarely realized by the host and that do not progress into invasive cancers, whereas in women long persisting infections apparently occur at substantially higher frequencies that can result in neoplastic transformation of the epithelial cells at the uterine transformation zone that finally may progress into invasive cancers. Interestingly this phenomenon seems to be restricted largely to few epithelial cells that occur at the transformation zone of the uterine cervix, whereas epithelial sites that are infected at comparable rates are substantially less prone to neoplastic transformation in comparison to epithelial cells of the uterine transformation zone.
During their normal life cycle HPVs infect first basal and parabasal cells of the epithelium. Several lines of arguments suggest that they first initiate a state of latent infection (Longworth and Laimins, 2004). During this the viral genome is replicated at very low copy numbers in single infected cells once the host cell divides, however no replication of the viral genome or even lytic infection is initiated at this stage. Due to technical limitations there is no true proof for the existence of this type of latent infection yet. It is anticipated rather from epidemiological studies that show that upon immunosuppression viral replication activity can be reactivated even if there is no evidence for de novo infections. These latent infections apparently can switch into active replicating infections. Here little viral gene expression activity is maintained in basal and parabasal cells, however, once the infected epithelial cells start to differentiate and reach the intermediate cell layers of the epithelium increasing gene expression is observed that triggers replication of the viral genomes in these differentiated cell layers. Interestingly, replication of viral genomes and expression of the viral early genes appears to be largely restricted to terminally differentiated cells that have irreversibly lost the capacity to proliferate and activated gene expression signatures of terminally differentiated epithelial cells that drive them into the pre-programmed pathway of differentiation and finally decay as squamous cell debris at the outer surface of the epithelium (Longworth and Laimins, 2004). Upon replication of the viral genomes in intermediate cells and once the epithelial cells reach during the normal differentiation processes the superficial cell layers, the viral genome again becomes re-programmed and expression of all early genes is stopped, whereas now full blown expression of the late genes L1 and L2 is observed that results in translation of the respective capsids proteins. These aggregate spontaneously to viral capsids in that the replicated viral genomes are included and finally the newly synthesized mature viral capsids are released from the decaying squamous cell debris at the outer surface of the epithelium and can re-initiate further infection cycles (FIG. 1). These findings suggest that the viral replication cycle is strictly linked to the differentiation processes of the normal epithelium. However, since the molecular events that trigger the differentiation of the epithelial cells, little or nothing is so far known about the molecular features that are involved in the regulation of the viral gene expression patterns that control this complex replication process.
Critical structures for the regulation of the viral gene expression as well as for the replication of the viral genomes are retained in a sequence element referred to as long control region (LCR) or upstream regulatory region or (URR). In this element various binding sites for important positive and negative transcription factors as well as the origin of replication are found (FIG. 2).
The papillomavirus E2 protein that is part of the several early expressed papillomavirus genes has substantial inhibitory functions on the expression of viral genes by binding to the viral promoter itself (Wells et al., 2000). It thus acts as a transcriptional repressor and prevents transcription of genes under control of the HPV URR element. This notion is based on a variety of molecular studies that have been performed in tissue culture models using non-differentiated epithelial cells grown in vitro or even cancer cell lines that retain the quasi non-differentiated phenotype of basal or parabasal cells. Expression of the E2 protein has been shown to result in down-regulation of the viral promoter and cessation of the expression of adjacent genes (Bouvard et al., 1994).
In cervical cancer cells, the papillomavirus genome is usually found to be integrated into host cell chromosomes (Pett and Coleman, 2007b). Whereas the integration site of the viral genome into the host cells genome appears to be randomly selected, is the viral genome of integrated viral DNA fragments usually preserved in a very peculiar manner. First of all are fragments that encompass the URR, and the E6 and E7 genes in all yet analyzed retain in an configuration that would still permit expression the E6 and E7 genes even from integrated viral genome copies, whereas the downstream located genes E2 and the late gene cassette is usually disconnected from the regulatory elements within the URR and hence functionally or even structurally inactivated (zur Hausen, 2002). Interestingly, in all HPV-associated carcinoma cells that have been investigated with this regard, expression of the viral E6 and E7 could be confirmed. As mentioned earlier, these genes confer important growth regulatory features to replicating cells through complex interactions with a number of host cell proteins that at least in part are involved in the regulation of the cell cycle, differentiation processes, and death cascades. The expression of these genes is a critical and sufficient prerequisite to induce and maintain neoplastic transformation of epithelial cells. If the expression of these genes is blocked in transformed cells these cells fall into cell cycle arrest, cease growth and eventually die (von Knebel et al., 1988).
In line with this observation is the open reading frame that encodes the E2 gene of HR-HPV types usually disrupted in HPV associated cancers. Re-introduction of functionally active E2 proteins into HPV-transformed cells therefore results in reversion of the neoplastic phenotype, cell cycle arrest and eventually cell death (Wells et al., 2000).
It is thus generally accepted among scientists in the field that disruption of the E2 ORF by integration into the host cell chromosomes is a critical if not absolutely essential step in the molecular cascade that finally results in malignant transformation of HPV-infected epithelial cells (Pett and Coleman, 2007a; Pett et al., 2006; Pett et al., 2004; Alazawi et al., 2002).
The tight association of the HPV replication cycle with features of epithelial differentiation suggests that the host cell milieu plays a critical role in the control of viral gene expression and replication (see FIG. 1). Several lines of evidence suggest that methylation and other forms of epigenetic regulation are involved in the gene expression and replication regulation of the viral genome. Most of these data are based on artificial tissue culture models. All models so far did not allow explaining the details of the differentiation dependent regulation of viral gene expression and replication.
Methylation of papillomavirus DNA was already described about 20 years ago (Burnett and Sleeman, 1984; Wettstein and Stevens, 1983) Its biological significance, however, in the regulation in papillomavirus gene expression control and the associated carcinogenic effects are still only poorly understood (List et al., 1994; Rost et al., 1993; Thain et al., 1996). It appears that CpG methylation in HPV-16 and HPV-18 genomes occurs more often in LCR regions and part of L1 ORF region than in any other parts of the virus genome in cervical cancer cell lines (Badal et al., 2004; Badal et al., 2003). Moreover, methylation particularly of the L1 gene appears to be associated with integration of the foreign viral DNA into the host cell genome during carcinogenesis and was thus suggested as potential biomarker for neoplastic conversion of HPV-infected cells (Kalantari et al., 2004; Turan et al., 2006). Further data also suggest that CpG methylation in E2BS prevents E2 protein binding in vitro (Thain et al., 1996) and modulates E2 protein function in cells in transcription activation (Bhattacharjee and Sengupta, 2006; Kim et al., 2003). However, DNA methylation in cancers is not restricted to HPV DNA. As outlined earlier, it is regarded as a very important feature, but rather occurs as a frequent event throughout the host genome (Esteller, 2007). The frequency of hypermethylation of many cellular genes has been found being increased significantly with increasing severity of neoplasia (Banno et al., 2007; Dong et al., 2001; Feng et al., 2005; Jeong et al., 2006; Kang et al., 2006; Lai et al., 2007; Lea et al., 2004; Reesink-Peters et al., 2004; Seng et al., 2007; Steenbergen et al., 2004; Virmani et al., 2001; Widschwendter et al., 2004).
A recent study investigated the presence or absence of methlytion of CpGs in E2 binding sites E2BS2 to 4 of HPV 16 in normal epithelium and cervical carcinoma cells (Bhattacharjee and Sengupta, 2006). These authors report that methylation was found in defined CpG islands that are located in the E2BS 2 to 4 proximal to the P97 promoter in the transformed tissues.
However, a clear consistent pattern that would explain distinct biological features with defined changes of the methylation pattern at specific sites could not been delineated in this study.
Kim et at (Kim et al., 2003) reported that hypomethylation was associated with in highly differentiated cell populations of an in vitro tissue culture model using W12 cells. In contrast, the HPV16 LCR from poorly differentiated, basal cell-like cells contained multiple methylated cytosines and were often methylated at E2BSs.
Moreover it has been disclosed in the prior art that human cancers frequently show altered patterns of DNA methylation, particularly at CpG islands. Methylation within islands has been shown to be associated with transcriptional repression of the linked gene. Genes involved in all facets of tumor development and progression can become methylated and epigenetically silenced. Re-expression of such silenced genes can lead to suppression of tumor growth or sensitization to anticancer therapies. Epigenetic agents that can reverse DNA methylation are now undergoing preclinical evaluation and clinical trials in cancer patients. The nucleoside inhibitors 5-azacytidine and decitabine have been tested in many phase I and II trials against many forms of cancer. However, the dose-limiting toxicity for both is myelosuppression, and the most commonly reported non-hematologic adverse effect was nausea and vomiting. Therefore a systemic therapy of cancers with demethylating agents is commonly causing severe side effects.
Specific DNA methylation inhibitors can be used for treating cosmetic and dermatologic conditions. It has been shown that 5 azacytidin inhibits both basal level TGFβ-induced collagen biosynthesis by normal human firoblasts. Collagen has a direct effect on scarring. Hence, inhibition of over-production of collagen in human skin results in inhibition of scar formation. Moreover, the combination of UV treatment and demethylation agent can be used for treatment of cancerous and pre-cancerous skin lesions.
The current invention relates to the finding that methylation of distinct, specific sequence elements within the papillomavirus genome are controlling the viral life cycle, replication and gene expression pattern. Moreover, it relates to the finding that a specific change of this methylation pattern is responsible for the inititiation of the neoplastic transformation induced by some papillomaviruses. This is particularly important for lesions of the mucosal epithelia of body cavities such as e.g. of the uterine cervix, vagina, vulva, anus and of the oropharyngeal tract. To overcome the unwanted and in part unbearable side effects associated with systemic therapies with demethylating agents the inventors found that especially for treatment of HPV related lesions and cancers of the mucosal epithelia of body cavities a topical treatment with demethylating agents may serve as a very effective approach to cure cancers without causing unwanted side effects to the patients.